1. Field of the Invention
The present invention is directed to the field of recombinant gene expression.
2. Discussion of the Related Art
There is a great demand for biologic molecules such as proteins, and particularly antibodies or antibody fragments, e.g., biologics that include the immunoglobulin Fc region.
Expression systems for the production of recombinant polypeptides are well-known in the state of the art and are described by, e.g., Marino M H (1989) Biopharm, 2: 18-33; Goeddel D V et al. (1990) Methods Enzymol 185: 3-7; Wurm F & Bernard A (1999) Curr Opin Biotechnol 10: 156-159. Polypeptides for use in pharmaceutical applications are preferably produced in mammalian cells such as Chinese Hamster Ovary (CHO) cells, NS0 cells, SP2/0 cells, COS cells, HEK cells, BHK cells, or the like. Various CHO-derived cell lines are particularly well-suited for industrial production of many different therapeutic biologic molecules. (E.g., Hu et al., U.S. Pat. No. 6,210,924 B1).
The essential elements of an expression vector used for this purpose are normally selected from a prokaryotic plasmid propagation unit, for example E. coli, comprising a prokaryotic origin of replication and a prokaryotic selection marker, optionally a eukaryotic selection marker, and one or more expression cassettes for the expression of the structural gene(s) of interest each comprising a promoter, a polynucleotide sequence encoding a polypeptide, and optionally a transcription terminator including a polyadenylation signal. For transient expression in mammalian cells a mammalian origin of replication, such as the SV40 Ori or OriP, can be included. As promoter a constitutive or inducible promoter can be selected. For optimized transcription a Kozak sequence may be included in the 5′ untranslated region. For mRNA processing, in particular mRNA splicing and transcription termination, mRNA splicing signals, depending on the organization of the structural gene (exon/intron organization), may be included as well as a polyadenylation signal. Expression of a gene is performed either in transient or using a stable cell line. However, the level of stable and high expression of a polypeptide in a production cell line is crucial to the overall process of the industrial production of recombinant polypeptides.
High cost and relatively poor yield have been limiting factors in the availability of biologic molecules and it has been a major challenge to develop robust processes that stably increase the yield of desirable biological molecules on an industrial scale. These and other benefits the present invention provides.